Genotypic and phenotypic diversity of Lactobacillus rhamnosus clinical isolates, their comparison with strain GG and their recognition by complement system

Abstract

Lactobacillus rhamnosus strains are ubiquitous in fermented foods, and in the human body where they are commensals naturally present in the normal microbiota composition of gut, vagina and skin. However, in some cases, Lactobacillus spp. have been implicated in bacteremia. The aim of the study was to examine the genomic and immunological properties of 16 clinical blood isolates of L. rhamnosus and to compare them to the well-studied L. rhamnosus probiotic strain GG. Blood cultures from bacteremic patients were collected at the Helsinki University Hospital laboratory in 2005-2011 and L. rhamnosus strains were isolated and characterized by genomic sequencing. The capacity of the L. rhamnosus strains to activate serum complement was studied using immunological assays for complement factor C3a and the terminal pathway complement complex (TCC). Binding of complement regulators factor H and C4bp was also determined using radioligand assays. Furthermore, the isolated strains were evaluated for their ability to aggregate platelets and to form biofilms in vitro. Genomic comparison between the clinical L. rhamnosus strains showed them to be clearly different from L. rhamnosus GG and to cluster in two distinct lineages. All L. rhamnosus strains activated complement in serum and none of them bound complement regulators. Four out of 16 clinical blood isolates induced platelet aggregation and/or formed more biofilms than L. rhamnosus GG, which did not display platelet aggregation activity nor showed strong biofilm formation. These findings suggest that clinical L. rhamnosus isolates show considerable heterogeneity but are clearly different from L. rhamnosus GG at the genomic level. All L. rhamnosus strains are still normally recognized by the human complement system.

Fig 1. Clustering analysis of strains by ortholog copy numbers.

The heat map rows represent centered and scaled ortholog copy numbers found in the strains. The dendrogram on the columns shows hierarchical clustering of the strains based on their ortholog copy numbers. Columns are annotated by each strain's BLAST nucleotide similarity to the L. rhamnosus GG genome.

Fig 2. A heatmap of phenotypic features of the L. rhamnosus strains measured in vitro.

Combined data represent average values or numbers of the strains detected in clusters A and B. NC equals negative control containing buffer in the assays, GAS group A streptococcus, NT not tested, asterisk* equals values that are representative from literature but not tested here for platelet aggregation [36] and biofilm formation [37], TCC equals terminal complement complex, FH factor H, C4bp complement C4b binding protein, CBA SpaC, SpaB and SpaA pilin subunits and fibron. bp equals fibronectin/fibrinogen binding protein. Bold and Italic letters equal significant value, p<0.05 for biofilm formation compared to L. rhamnosus GG (GG). Color intensities indicate the level of the values: green for complement activation measured as C3a and TCC, red for binding of complement regulators, blue for platelet aggregation, purple for biofilm formation and light green express the presence of SpaCBA pilus determined by Western blot using anti-SpaA antibody and fibronectin/fibrinogen binding protein at genome.

Fig 3. Genomic organization of polysaccharide clusters in L. rhamnosus strains.

A) EPS gene cluster of L. rhamnosus GG is found from L. rhamnosus strains of cluster A and strain Lrh20. Insertion sequence (IS)-like elements differ between the strains. B) The L. rhamnosus strains of cluster B have differences at sequences encoding genes in the EPS gene cluster compared to L. rhamnosus GG. The genes similar to the EPS gene cluster of L. rhamnosus GG are named in the schematic illustration in the upper part (B). These regions include multiple genes coding IS-like elements such as IS5, IS66 and DDE transposases in the cluster B. The strains in cluster B have also other type of EPS/CPS gene cluster based on BLAST annotation results shown at the bottom (B). It consists of 19 or 20 genes which are annotated as 1. peptidoglycan N-acetylglucosamine deacetylase (EC 3.5.1.104), 2. hypothetical protein (FIG00750667), 3. capsular polysaccharide biosynthesis protein, 4. hypothetical protein (FIG00754398), 5. tyrosine-protein kinase transmembrane modulator EpsC, 6. putative O-unit flippase, 7. glycosyl transferase, group 0, 8. glycosyl transferase family protein, 9. hypothetical protein (FIG00752018), 10. mobile element protein, 11. lysozyme M1 (1,4-beta-N-acetylmuramidase), 12. hypothetical protein (FIG00747485), 13. uncharacterized conserved protein, similar to IcaC of Staphylococcus; YHJR B.subtilis family, 14. glycosyltransferase, 15. UDP-N-acetylglucosamine 2-epimerase (EC 5.1.3.14), 16. hypothetical protein (FIG00750997), 17. lipopolysaccharide synthesis sugar transferase, 18. polysaccharide biosynthesis protein CpsM, 19. putative N-acetylgalactosaminyldiphosphoundecaprenol and 20. UDP-glucose 4-epimerase (EC 5.1.3.2). * = Transposase, IS150/IS3 present (only in Lrh11), ↑ = site of point mutations in the gene encoding lysozyme M1(1,4-beta-N-acetylmuramidase) (present in Lrh13 and Lrh28). The illustration of EPS cluster in 3A is modified after [46].

Fig 4. Immunological properties of L. rhamnosus GG, clinical isolates and pathogens towards the human host based on in vitro data.

Strains Lrh7, Lrh8, Lrh9, Lrh14, Lrh20, Lrh46, Lrh47, Lrh38 and Lrh39 displayed comparable properties as observed in L. rhamnosus GG. All these strains activate the complement in serum. Strains Lrh13, Lrh30 and Lrh32 also induced more biofilm formation more than GG. Strains Lrh11, Lrh18 and Lrh22 were aggregating with platelets. Strain Lrh28 was doing both causing platelet aggregation and biofilm formation. The pathogenic group A Streptococcus strain emm8 induced complement activation, but it could bind complement regulator C4bp to evade complement. The pathogenic strain st369 could bind complement regulators C4bp and factor H and did not induce complement activation. A similar type of GAS strain has been shown to cause platelet aggregation [36] and biofilm formation [37]. The bolded strains have SpaCBA pili similar to GG.